WO2007059250A2 - Plant-based formulations for improving liver health - Google Patents

Plant-based formulations for improving liver health Download PDF

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Publication number
WO2007059250A2
WO2007059250A2 PCT/US2006/044420 US2006044420W WO2007059250A2 WO 2007059250 A2 WO2007059250 A2 WO 2007059250A2 US 2006044420 W US2006044420 W US 2006044420W WO 2007059250 A2 WO2007059250 A2 WO 2007059250A2
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WIPO (PCT)
Prior art keywords
extract
liver
formulation
root
notoginseng
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PCT/US2006/044420
Other languages
French (fr)
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WO2007059250A3 (en
Inventor
Chioma Jane Ikonte
Michael Huang
Cherie L. Hacker
Silvia R. Dacosta
Paul D. Johnson
Amitabh Chandra
Christine M. Paganelli
Ruo G. Huang
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Access Business Group International Llc
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Priority to JP2008541325A priority Critical patent/JP2009515987A/en
Priority to KR1020087011534A priority patent/KR101350053B1/en
Priority to CN2006800506996A priority patent/CN101415433B/en
Publication of WO2007059250A2 publication Critical patent/WO2007059250A2/en
Publication of WO2007059250A3 publication Critical patent/WO2007059250A3/en
Priority to HK09107660.5A priority patent/HK1129839A1/en

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18Magnoliophyta (angiosperms)
    • A61K36/185Magnoliopsida (dicotyledons)
    • A61K36/79Schisandraceae (Schisandra family)
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18Magnoliophyta (angiosperms)
    • A61K36/185Magnoliopsida (dicotyledons)
    • A61K36/21Amaranthaceae (Amaranth family), e.g. pigweed, rockwort or globe amaranth
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18Magnoliophyta (angiosperms)
    • A61K36/185Magnoliopsida (dicotyledons)
    • A61K36/25Araliaceae (Ginseng family), e.g. ivy, aralia, schefflera or tetrapanax
    • A61K36/258Panax (ginseng)
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18Magnoliophyta (angiosperms)
    • A61K36/185Magnoliopsida (dicotyledons)
    • A61K36/28Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18Magnoliophyta (angiosperms)
    • A61K36/185Magnoliopsida (dicotyledons)
    • A61K36/31Brassicaceae or Cruciferae (Mustard family), e.g. broccoli, cabbage or kohlrabi
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18Magnoliophyta (angiosperms)
    • A61K36/185Magnoliopsida (dicotyledons)
    • A61K36/36Caryophyllaceae (Pink family), e.g. babysbreath or soapwort
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18Magnoliophyta (angiosperms)
    • A61K36/185Magnoliopsida (dicotyledons)
    • A61K36/48Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A61K36/488Pueraria (kudzu)
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18Magnoliophyta (angiosperms)
    • A61K36/185Magnoliopsida (dicotyledons)
    • A61K36/53Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18Magnoliophyta (angiosperms)
    • A61K36/185Magnoliopsida (dicotyledons)
    • A61K36/81Solanaceae (Potato family), e.g. tobacco, nightshade, tomato, belladonna, capsicum or jimsonweed
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18Magnoliophyta (angiosperms)
    • A61K36/88Liliopsida (monocotyledons)
    • A61K36/896Liliaceae (Lily family), e.g. daylily, plantain lily, Hyacinth or narcissus
    • A61K36/8965Asparagus, e.g. garden asparagus or asparagus fern
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention is directed to unique formulations of plant-based extracts that work synergistically to aid in good liver health. More specifically, the present invention relates to unique formulations of plant-based extracts that assist in protecting the liver from alcohol and chemically induced damage.
  • the liver is one of the hardest working organs in the body. Good liver function is important for balanced hormone levels, weight control and maintenance, cholesterol levels, skin health and general health.
  • the liver serves as the body's clearing house and is responsible for the metabolism of a number of substances, including alcohol, and plays an important role in the detoxification of toxins in the body.
  • Phase II enzymes are part of this detoxification process because they aid in the removal of potential carcinogens from the body.
  • the liver is under constant attack and prone to damage from environmental toxins, impurities, alcohol, prescription and over-the-counter drugs.
  • hepatotoxicants such as carbon tetrachloride, nitrosamines, and polycyclic aromatic hydrocarbons are metabolically activated by liver enzymes to form reactive, toxic metabolites that cause injury to the liver in humans.
  • liver enzymes to form reactive, toxic metabolites that cause injury to the liver in humans.
  • formulations of plant-based extracts that aid in protecting the liver against alcohol and carbon tetrachloride insults would be useful.
  • plant-based formulations that induce phase II enzymes responsible for detoxifying the liver would be useful.
  • the present invention is directed to unique formulations that improve liver health by working to protect the liver from carbon tetrachloride and alcohol insults.
  • Formulations of the present invention have shown strong protective abilities on human liver cells as measured by indices such as 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide
  • phase II enzyme induction assay measures a sample's ability to induce quinone reductase (a phase II enzyme) which is indicative of detoxification events.
  • the present invention provides a formulation for improving liver health that includes wasabi root fiber powder, artichoke leaf extract, asparagus extract, kudzu root extract, oregano extract, schisandra berry extract, notoginseng (ethanol extract of Panax notoginseng root), sanchi (water extracts from Panax notoginseng root), herbs ⁇ Pueraria omeiensis), spinach dehydrate, or combinations thereof.
  • the present invention provides a method of protecting the liver from carbon tetrachloride ("CCl 4 ”) insults by providing wasabi root fiber powder, artichoke leaf extract, asparagus extract, kudzu root extract, oregano extract, schisandra berry extract, notoginseng (ethanol extract of Panax notoginseng root), sanchi (water extracts from Panax notoginseng root), herbs (Pueraria omeiensis), spinach dehydrate, or combinations thereof.
  • CCl 4 carbon tetrachloride
  • the present invention provides methods of protecting the liver from alcohol insults by providing wasabi root fiber powder, artichoke leaf extract, asparagus extract, kudzu root extract, oregano extract, schisandra berry extract, notoginseng (ethanol extract of Panax notoginseng root), sanchi (water extracts from Panax notoginseng root), herbs (Pueraria omeiensis), spinach dehydrate, or combinations thereof.
  • the present invention provides methods of inducing phase II enzymes by providing wasabi root fiber powder, artichoke leaf extract, asparagus extract, kudzu root extract, oregano extract, schisandra berry extract, notoginseng (ethanol extract of Panax notoginseng root), sanchi (water extracts from Panax notoginseng root), herbs (Pueraria omeiensis), spinach dehydrate, or combinations thereof.
  • Figure 1 is a bar graph showing the effect of various formulations in protecting the liver from ethanol insults as measured by MTT.
  • Figure 2 is a bar graph showing the effect of various formulations in protecting the liver from ethanol insults as measured by LDH.
  • Figure 3 is a bar graph showing the effect of various formulations in protecting the liver from CCl 4 insults as measured by MTT.
  • Figure 4 is a bar graph showing the effect of various formulations in protecting the liver from CCl 4 insults as measured by LDH. •
  • the present invention is based on the surprising discovery that unique combinations of the following ingredients, described more folly in Table 1, improve liver health: wasabi root fiber powder, artichoke leaf extract, asparagus extract, kudzu root extract, oregano extract, schisandra berry extract, notoginseng (ethanol extract of Panax notoginseng root), sanchi (water extracts from Panax notoginseng root), herbs (Pueraria omeiensis), and spinach dehydrate.
  • the formulations of the present invention improve liver health by protecting the liver against alcohol and carbon tetrachloride insults. Additionally, the formulations improve liver health by inducing phase II enzymes. Phase II enzymes are responsible for removing potential carcinogens by aiding in their removal from the body.
  • Table 2 illustrates representative daily amounts of the ingredients that can be included in the supplement.
  • Example 1 The following are illustrative examples of formulations made into tablets according to this invention and it should be understood that they do not limit the scope of the invention.
  • Example 1 The following are illustrative examples of formulations made into tablets according to this invention and it should be understood that they do not limit the scope of the invention.
  • tabletted formulations can be manufactured according to typical methods known in the industry. For example, wasabi root fiber powder, artichoke leaf extract, schizandra berry extract, notoginseng and spinach dehydrate are passed through a SWECO separator equipped with a 20 mesh screen into a 100 cubic foot PK blender. Microcrystalline cellulose is added to the blend in the PK blender. The ingredients are blended for ten minutes. Cellulose gum and silicon dioxide are passed through a SWECO separator equipped with a 20 mesh screen directly into the 100 cubic foot PK blender. The ingredients are blended for ten minutes. Next, stearic acidis passed through a SWECO separator equipped with a 20 mesh screen directly into the 100 cubic foot PK blender. The mixture is blended for an additional five minutes. The resulting mixture is discharged into totes or supersacks, and compressed into tablets.
  • wasabi root fiber powder, artichoke leaf extract, schizandra berry extract, notoginseng and spinach dehydrate are passed through a SWECO separator equipped with a 20 mesh screen into a 100
  • Formulations of the present invention may be formulated in an acceptable carrier and may be prepared, packaged, and labeled for promoting health, liver function, protecting against alcohol and/or chemical insults to the liver, and/or inducing phase II enzymes to promote healthy liver function.
  • the formulations of the present invention and their acceptable carriers may be formulated for oral administration in the form of a pill, tablet, dried or powdered product for reconstitution with water or other suitable vehicle before use, bar, food, solution, syrup, suspension, beverage, lozenge, etc.
  • the formulations of the present invention may also be parenterally administered or administered by inhalation or insufflation (either through the mouth or nose).
  • Liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).
  • suspending agents e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats
  • emulsifying agents e.g., lecithin or acacia
  • non-aqueous vehicles e.g., almond oil, oily esters, or fractionated vegetable oils
  • preservatives e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid
  • formulations of the present invention may be water-based, milk-based, tea-based,
  • Formulations of the present invention may also be orally administered in the form of a solid prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose; fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., cellulose gum, potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate).
  • binding agents e.g., pregelatinized maize starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose
  • fillers e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate
  • lubricants e.g., magnesium stearate, talc or silica
  • disintegrants
  • Formulations of the present invention that are orally administered can further comprise thickeners, including xanthum gum, carboymethyl-cellulose, carboxyethylcellulose, hydroxypropylcellulose, methylcellulose, microcrystalline cellulose, starches, dextrins, fermented whey, tofu, maltodextrins, polyols, including sugar alcohols (e.g., sorbitol and mannitol), carbohydrates (e.g. lactose), propylene glycol alginate, gellan gum, guar, pectin, tragacanth gum, gum acacia, locust bean gum, gum arabic, gelatin, as well as mixtures of these thickeners.
  • thickeners including xanthum gum, carboymethyl-cellulose, carboxyethylcellulose, hydroxypropylcellulose, methylcellulose, microcrystalline cellulose, starches, dextrins, fermented whey, tofu, maltodextrins, polyols
  • Orally administered formulations of the present invention can contain an effective amount of one or more sweeteners, including carbohydrate sweeteners and natural and/or artificial no/low calorie sweeteners.
  • the amount of the sweetener used in the formulations of the present invention will vary, but typically depends on the type of sweetener used and the sweetness intensity desired.
  • the compounds may also be a formulated as a sustained and/or timed release formulation.
  • the formulations must be maintained above some minimum therapeutic dose to be effective.
  • Common timed and/or controlled release delivery systems include, but are not be restricted to, starches, osmotic pumps, or gelatin micro capsules.
  • the formulations may, if desired, be presented in a pack or dispenser device which may comprise one or more unit dosage forms comprising a formulation of the present invention.
  • the pack may for example comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • compositions can be prepared by methods and techniques that will be well understood by those of skill in the art and may include the use of additional ingredients in producing tablets, capsules, or liquid dosage forms.
  • the dose, and dose frequency will vary according to the age, body weight, condition and response of the individual consumer or patient, and the particular formulation of the present invention that is used.
  • phase II enzyme induction testing was also conducted to determine if a material had detoxification properties.
  • the phase II enzyme induction assay measures a sample's ability to induce quinone reductase (a phase II enzyme) which is indicative of detoxification events.
  • Phase II enzymes are responsible for removing potential carcinogens by aiding in their removal from the body. Broccoli is known as a good phase II enzyme inducer because of its sulforaphane content.
  • Pure sulforaphane has activity at 10 6 U/g, while broccoli has reported activity from 4000 - 74;000 U/g (dependent on variety, form, and/or extraction conditions). Activity above 30,000 U/g is considered excellent. Activity less than 5000 U/g is considered minimal. Most materials that are considered good phase II enzyme inducers will have activity between 15,000-30,000 U/g.
  • Tables 3 and 4 summarize the EC-50 values of all materials tested. Table 3 is specifically for results of the ethanol protection, and Table 4 for carbon tetrachloride. Table 5 gives the results of the phase II enzyme induction assay.
  • Table 4 EC-50 results of the MTT and LDH assays for CC14 protection assessment.
  • the second assay is the MTT assay, which measures the reduction of a yellow tetrazolim salt into an insoluble purple formazen product by the mitochondria of viable cells. Following an incubation with the MTT solution, isopropanol is added to solubilize the colored crystals. The amount of color produced is directly proportional to the number of viable cells.
  • MTT MTT assay
  • stock sample solutions are made in acetonitrile, then diluted in cell culture media for testing.
  • Treatment of Hepalclc7 cells (murine hepatoma cell line) is done by adding 150 ⁇ L of sample to each of three wells, in a 96-well microtiter plate.
  • induction activity of quinone reductase is established by measuring the NADPH-dependent, menadiol-mediated reduction of MTT. Activity is reported as inducer units per gram of fresh weight of material, where one unit of inducer activity is defined as the amount of inducer required to double the quinone reductase specific activity of Hepalclc7 cells.
  • liver detoxification ingredients were submitted for bioassay testing to try to predict their protective ability against alcohol and chemically induced liver damage. These blends were tested alongside two products already on the market for liver health - NUTRILITE® Milk Thistle and Dandelion and China's King Drink. To assess this, human liver cells were treated with the sample and an insult, and cell viability was measured using two different assays. Alcohol damage was mimicked using 2.5% ethanol as the insult. CCl 4 at 0.2% was used for the chemical insult. These concentrations were chosen because they were the concentrations that produced 20% cell death in preliminary experiments, and would therefore not cause the cells to undergo irreversible cell death/damage.
  • the blends that showed the greatest efficacy against CCl 4 liver cell damage were 8523- 25-CI (Example 1), 8523-27-CI (Example 8), 8523-28-CI (Example 2), and 8523-30-CI (Example 3).
  • Other blends that performed well were 8523-20-CI, 8523-22-CI, 8523-24-CI, 8523-26-CI and 8523-31 -CI. None of the blends showed efficacy at ⁇ 10 ⁇ g/mL against ethanol liver cell damage.
  • the control products (NUTRILITE milk thistle and King Drink) also did not achieve EC-50 values of ⁇ 10 ⁇ g/mL.
  • the blends that had EC-50 values >100 ⁇ g/mL were 8523-28-CI (Example 2), 8523-30-CI (Example 3), 8523-31 -CI (Example 4) and 8523-32-CI (Example 5).
  • Tables 6-9 summarize the results of all the samples tested. These results are also seen in Figures 1-4.
  • Table 9 EC-50 results of the LDH assays for CC14 protection assessment.
  • phase II enzyme induction testing was also conducted to determine if a material had detoxification properties.
  • the phase II enzyme induction assay measures a sample's ability to induce quinone reductase (a phase II enzyme) which is indicative of detoxification events.
  • Broccoli is known as a good phase II enzyme inducer because of its sulforaphane content. Pure sulforaphane has activity at 10 6
  • Table 10 gives the results of the phase II enzyme induction assay for all samples tested.
  • Table 10 Phase II Enzyme (Quinone Reductase) Induction Assay Results
  • Experimental Stock sample solutions are made in DMSO, then diluted in cell culture media for testing.
  • Treatment of HepG2 cells is done by adding 100 ⁇ L of sample to each of three wells of a 96-well microtiter plate. After a 4 hour incubation, the insult is added (2.5% ethanol or 0.2% carbon tetrachloride) and an additional overnight incubation period is conducted. The next day, cell viability is measured using two different assays. First, using the CYTOTOX-ONETM Homogenous Membrane Integrity Assay by Promega, the number of non- viable cells is estimated by measuring the release of LDH into the media. LDH leaks out of the cell when the cell membrane is compromised.
  • the second assay is the MTT assay, which measures the reduction of a yellow tetrazolim salt into an insoluble purple formazen product by the mitochondria of viable cells. Following an incubation with the MTT solution, isopropanol is added to solubilize the colored crystals. The amount of color produced is directly proportional to the number of viable cells. Protection is determined by first calculating a percent toxicity of each well (1- experimental/negative control), after averaging the three replicates. Percent protection is then calculated by the following: (%Toxicity Positive Control - % Toxicity Sample)/% Toxicity Positive Control, the positive control being either 2.5% ethanol or 0.2% carbon tetrachloride. The concentration that exhibits 50% protection (EC-50) can then be assessed. For the purpose of this experiment, this was categorized as either ⁇ 1, 1, 1-10, 10, 10-100, 100 or >100 ⁇ g/mL.
  • phase II enzyme induction assay stock sample solutions are made in acetonitrile, then diluted in cell culture media for testing.
  • Treatment of Hepalclc7 cells is done by adding 150 ⁇ L of sample to each of three wells, in a 96-well microtiter plate. After 48 hours incubation, induction activity of quinone reductase is established by measuring the NADPH-dependent, menadiol-mediated reduction of MTT. Activity is reported as inducer units per gram of fresh weight of material, where one unit of inducer activity is defined as the amount of inducer required to double the quinone reductase specific activity of Hepal cl c7 cells.
  • Clinical testing can be conducted to confirm the efficacy of the formulations on liver health. It is expected that the formulations will improve liver health by protecting the liver from chemical and alcohol insults. A protocol for such testing follows. PROTOCOL 1 : CCl 4 LIVER INJURY MODEL 1.1 Principles.
  • CCl 3 - When CCl 4 is activated by microsomal enzyme of liver, trichloromethane free radicals (CCl 3 -) are formed. Covalent combination of this free radical with protein results in impairment of protein synthesis and disorder of lipid catabolism, causing accumulation of triglyceride (TG) in liver cells. CCl 3 - also can combine with O 2 rapidly to form trichloromethane peroxide free radicals (CCl 3 O 2 -), leading to lipid peroxidation which causes degenerative injury of cell membrane, leakage of enzymes and various types of pathological changes of cells and even necrosis. 1.2 Experimental animals.
  • mice of single sex Each group consists of 8-12 rats (180-22Og) or 10- 15 mice (18-22g).
  • Experimental methods and procedures 1.3.1 Dosage groups and duration of administration of the test sample.
  • the dosage of one of the dosage groups is 10 times (mice) or 5 times (rats) the recommended human dosage.
  • CCl 4 (analytically pure) is used to form liver injury model.
  • the method of forming model can use intragastric administration or intraperitoneal injection.
  • the concentration of CCl 4 for intragastric administration in mice is 1%.
  • CCl 4 is diluted with edible vegetable oil and the dosage for intragastric administration is 5mL/kg BW (the dosage in terms of CCl 4 is 80mg/kg BW).
  • the concentration of CCl 4 for intragastric administration in rats is 2%-3% and the dosage is 5mL/kg BW (the dosage in terms of CCl 4 is 160-240mg/kg BW).
  • Positive control group and solvent control group may be set if necessary.
  • the duration of administration of the test sample is 30 days and can be prolonged to 45 days if necessary.
  • test sample is given intragastrically. If this is impossible, the test sample can be mixed into the feed or drinking water and the feed intake or water drunk is recorded.
  • the animals of the experimental group are given intragastrically the test sample daily, while those of the blank control group and the model control group are given distilled water. The animals are weighed twice a week for adjusting the dosage of the test sample. On the eve of day 30 of the experiment, the animals of various groups fast for 16h. The animals of the model group and various test sample groups are given intragastrically single dose of CCl 4 , while those of the blank control group are given vegetable oil. The animals of the experimental group continue to receive the test sample until the end of the experiment (the interval between administration of the test sample and CCl 4 is over 4h). After giving CCl 4 , the animals are sacrificed 24h or 48h later according to the actual conditions. Blood is taken and serum is separated for measuring ALT and AST. Liver is taken for histopathological examination.
  • ALT Serum glutamate-pyruvate transaminase
  • AST glutamic-oxaloacetic transaminase
  • histopathological examination of liver 1.4 Measurement of ALT and AST. 1.4.1 Measuring method.
  • Full-automatic biochemical analyzer or Reitman-Frankel method (reagent kit) can be selected. 1.4.1 Data treatment and result assessment.
  • Variance analysis is used, but variance homogeneity test should be performed first according to the procedures of variance analysis. If the variance is homogeneous, F value is calculated. If F value is ⁇ F o .o 5 , the conclusion is that the difference between means of different groups is not significant. If F value is > Fo. O5 and P is ⁇ 0.05, the method of paired comparison of means between several experimental groups and one control group is used for statistical analysis. For data with abnormal distribution or variance inhomogeneity, appropriate conversion of variables is performed and the converted data are used for statistical analysis after the requirement of normal or variance homogeneity is fulfilled. If the purpose of normal or variance homogeneity is still not achieved after conversion of variables, rank test is used for statistical analysis.
  • ALT and AST of the test sample group are different significantly from those of the model control group, the results of ALT and AST can be assessed as positive respectively. 1.5 Histopatholofiical changes of liver, diagnostic criteria and result assessment. 1.5.1 Experimental materials.
  • the left lobe of rat liver is fixed with 10% formalin.
  • the liver tissue is taken from the cross section of middle part of the left lobe of liver for routine preparation of pathological section (paraffin embedding, H.E. strain). 1.5.2 Microscopic examination.
  • the pathological changes of cells are recorded beginning from the visual field of one end of the liver.
  • the degenerative changes of central liver cells of the lobe and necrosis of a few cells can be seen.
  • the main types of pathological changes are ballooning degeneration, fatty degeneration, condensation of cytoplasm, hydropic degeneration and necrosis of liver cells, etc.
  • Liver cells with ballooning degeneration account for 1/4 of whole visual field 1 point
  • liver cells Fatty degeneration of liver cells: (distinctly demarcated fat drop vacuoles appear in cytoplasm of liver cells)
  • Liver cells with fatty degeneration account for 1/4 of whole visual field 1 point
  • Liver cells with condensation of cytoplasm account for 3/4 of whole visual field 3 points
  • Liver cells with condensation of cytoplasm account for whole visual field 4 points
  • necrosis necrosis
  • liver cells including ballooning degeneration, fatty degeneration, cytoplasm condensation , hydropic degeneration and necrosis of liver cells
  • the necrosis of liver cells in any dosage group of test sample is alleviated as compared with that in the model control group with significant difference and other types of pathological changes are alleviated significantly or have no significant difference as compared with those in the model control group, the results of animal pathological experiment can be assessed as positive.
  • ethyl alcohol After taking large amounts of ethyl alcohol, massive dehydroxylation catalyzed by ethanol dehydrogenase causes disorder of tricarboxylic acid cycle and weakening of oxidation of fatty acid, thereby influencing fat metabolism and precipitation of fat in liver cells. At the same time, ethyl alcohol can activate oxygen molecules and cause production of oxygen free radicals, leading to lipid peroxidation of liver cell membrane and depletion of reduced glutathione in body.
  • mice or rats of single sex Each group consists of 8-12 rats (180-22Og) or 10- 15 mice (18-22g).
  • Three dosage groups, one blank control group and one model control group are set.
  • the dosage of one of the dosage groups is 10 times (mice) or 5 times (rats) the recommended human dosage.
  • a positive control group may be set if necessary.
  • Absolute ethyl alcohol (analytically pure) is used to form model of liver injury.
  • the concentration of absolute ethyl alcohol is 50% (diluted with distilled water) and the dosage for intragastric administration to mice is 12-14mL/kg BW (equivalent to ethyl alcohol 6000-7000mg/kg BW).
  • the duration of administration of the test sample is 30 days and can be prolonged to 45days if necessary.
  • test sample is given intragastrically. If intragastric administration is impossible, the test sample can be mixed in feed or drinking water, and the feed intake and drinking water drunk of each animal is recorded. 2.3.3 Experimental procedures.
  • the animals of the test sample groups are given intragastrically the test sample every day and those of the blank control group and model control group are given distilled water.
  • the animals are weighed twice weekly and the dose of the test sample is adjusted according to body weight.
  • a single dose of 50% ethyl alcohol 12mL/kg BW is given to the animals of the model control group and three dosage groups, while the animals of the blank control group are given distilled water.
  • MDA Malondialdehyde
  • GSH reduced glutathione
  • TG triglyceride
  • MDA is one of the final products of peroxidation of lipids of cell membrane. Measuring MDA content can estimate indirectly the degree of lipid peroxidation. When MDA and thiobarbital are heated together in acidic condition, pink-colored complex is formed and its absorption peak is at 535nm, from which the MDA content can be measured.
  • Tissue homogenate sample certain quantity of the organ needed is rinsed with normal saline, wiped to dry, weighed, minced and put into homogenizer. 0.2 M phosphate buffer solution is added and the mixture is homogenized at 2000r/min for 10s. The centrifugation is repeated 3 times with 30s intervals to form 5% tissue homogenate (WfV). The homogenate is centrifugalized at 3000r/min for 5-10min and the supernatant is taken for measurement.
  • Lipid peroxide content B - A B - A 1 1
  • variance homogeneity test should be performed first according to the procedures of variance analysis. If the variance is homogeneous, F value is calculated. If F value is ⁇ F 0 . 05 , the conclusion is that the difference between means of different groups is not significant. IfF value is > Fo.os and P is ⁇ 0.05, the method of paired comparison of means between several experimental groups and one control group is used for statistical analysis. For data with abnormal distribution or variance inhomogeneity, appropriate conversion of variables is performed and the converted data are used for statistical analysis after the requirement of normal or variance homogeneity is fulfilled. If the purpose of normal or variance homogeneity is still not achieved after conversion of variables, rank test is used for statistical analysis.
  • NaH 2 PO 4 1.076g Distilled water ad 100OmL. Small amount of HCl and NaOH are used to regulate pH 7.0.
  • Standard solution Reduced GSH 15.4mg is weighed and sodium azide buffer solution is added to 5OmL to make the final concentration lmmol/L.
  • the solution is prepared just before use.
  • Normal saline 5mL is added to liver 0.5g.
  • the mixture is well ground to form fine thick liquid (10% liver homogenate).
  • 4% sulfosalicylic acid 0.5mL is added to the homogenate 0.5mL.
  • the mixture is centrifugalized at 3000rpm for lOmin at room temperature and the supernatant is the sample.
  • the mixture is mixed, laid aside for lOmin at room temperature and its absorbance is measured at 412nm.
  • GSH content ( ⁇ mol/L) 0 100 200 300 400 500
  • the data are analyzed with variance analysis, but variance homogeneity test is performed first according to the procedures of variance analysis. If the variance is homogeneous, F value is calculated. If F value is ⁇ F o .o 5 , the conclusion is that the difference between means of different groups is not significant. If F value is > F o .o 5 and P is ⁇ 0.05, the method of paired comparison of means between several experimental groups and one control group is used for statistical analysis. For data with abnormal distribution or variance inhomogeneity, appropriate conversion of variables is performed and the converted data are used for statistical analysis after the requirement of normal or variance homogeneity is fulfilled. If the purpose of normal or variance homogeneity is still not achieved after conversion of variables, rank test is used.
  • Triglyceride measurement reagent kit (glycerophosphoric acid oxidase peroxidase method) is used to measure the triglyceride content in 10% liver homogenate. Same as the method of measuring serum triglyceride, equal amount of 10% liver homogenate is used instead of serum and the measurement is performed according to the description of operation. The result of measurement is expressed as mmol/g liver weight. 2.6.2 Data treatment and result assessment.
  • ⁇ 0.05 the method of paired comparison of means between several experimental groups and one control group is used for statistical analysis.
  • appropriate conversion of variables is performed and the converted data are used for statistical analysis after the requirement of normal or variance homogeneity is fulfilled. If the purpose of normal or variance homogeneity is still not achieved after conversion of variables, rank test is used for statistical analysis. Assessment of results
  • Cross section at middle part of left lobe of liver is performed for taking examination material. Frozen section is made and stained with Sudan III staining.
  • the fat drops in liver cells are sporadic and scarce 0 points
  • liver cells containing fat drops do not exceed 1/4 1 point
  • liver cells containing fat drops do not exceed 1/2 2 points
  • liver cells containing fat drops do not exceed 3/4 3 points
  • the liver tissue is almost replaced by fat drops 4 points
  • Variance analysis is used, but variance homogeneity test should be performed first according to the procedures of variance analysis. If the variance is homogeneous, F value is calculated. If F value. is ⁇ Fo.os, the conclusion is that the difference between means of different groups is not significant. IfF value is > F 0 . 05 and P is ⁇ 0.05, the method of paired comparison of means between several experimental groups and . one control group is used for statistical analysis. For data with abnormal distribution or variance in homogeneity, appropriate conversion of variables is performed and the converted data are used for statistical analysis after the requirement of normal or variance homogeneity is fulfilled. If the purpose of normal or variance homogeneity is still not achieved after conversion of variables, rank test is used for statistical analysis.
  • test sample would be assessed as assisting in protection against alcoholic liver injury:

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Abstract

The present invention is directed to plant based formulations for improving liver health by protecting the liver from alcohol and chemical insults and/or by inducing phase II enzymes. Formulations according to the present invention include wasabi root fiber powder, artichoke leaf extract, asparagus dehydrate, kudzu root extract, oregano extract, schisandra berry extract, notoginseng (ethanol extract of Panax notoginseng root), sanchi (water extracts from Panax notoginseng root), Gegen root extract (Pueraria omeiensis), spinach dehydrate, or combinations thereof.

Description

PLANT-BASED FORMULATIONS FOR IMPROVING LIVER HEALTH
BACKGROUND OF THE INVENTION
The present invention is directed to unique formulations of plant-based extracts that work synergistically to aid in good liver health. More specifically, the present invention relates to unique formulations of plant-based extracts that assist in protecting the liver from alcohol and chemically induced damage.
The liver is one of the hardest working organs in the body. Good liver function is important for balanced hormone levels, weight control and maintenance, cholesterol levels, skin health and general health. The liver serves as the body's clearing house and is responsible for the metabolism of a number of substances, including alcohol, and plays an important role in the detoxification of toxins in the body. Phase II enzymes are part of this detoxification process because they aid in the removal of potential carcinogens from the body. As a result of its function in the body, the liver is under constant attack and prone to damage from environmental toxins, impurities, alcohol, prescription and over-the-counter drugs. Many hepatotoxicants such as carbon tetrachloride, nitrosamines, and polycyclic aromatic hydrocarbons are metabolically activated by liver enzymes to form reactive, toxic metabolites that cause injury to the liver in humans. Thus, formulations of plant-based extracts that aid in protecting the liver against alcohol and carbon tetrachloride insults would be useful. Additionally, plant-based formulations that induce phase II enzymes responsible for detoxifying the liver would be useful.
SUMMARY OF THE INVENTION
The present invention is directed to unique formulations that improve liver health by working to protect the liver from carbon tetrachloride and alcohol insults. Formulations of the present invention have shown strong protective abilities on human liver cells as measured by indices such as 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide
("MTT"), which is a tetrazolium salt, and lactase dehydrogenase ("LDH"). In addition, these extracts and their combinations show strong phase II enzyme induction activity. The phase II enzyme induction assay measures a sample's ability to induce quinone reductase (a phase II enzyme) which is indicative of detoxification events.
Accordingly, in one embodiment, the present invention provides a formulation for improving liver health that includes wasabi root fiber powder, artichoke leaf extract, asparagus extract, kudzu root extract, oregano extract, schisandra berry extract, notoginseng (ethanol extract of Panax notoginseng root), sanchi (water extracts from Panax notoginseng root), Gegen {Pueraria omeiensis), spinach dehydrate, or combinations thereof.
In another embodiment, the present invention provides a method of protecting the liver from carbon tetrachloride ("CCl4") insults by providing wasabi root fiber powder, artichoke leaf extract, asparagus extract, kudzu root extract, oregano extract, schisandra berry extract, notoginseng (ethanol extract of Panax notoginseng root), sanchi (water extracts from Panax notoginseng root), Gegen (Pueraria omeiensis), spinach dehydrate, or combinations thereof. / In yet another embodiment, the present invention provides methods of protecting the liver from alcohol insults by providing wasabi root fiber powder, artichoke leaf extract, asparagus extract, kudzu root extract, oregano extract, schisandra berry extract, notoginseng (ethanol extract of Panax notoginseng root), sanchi (water extracts from Panax notoginseng root), Gegen (Pueraria omeiensis), spinach dehydrate, or combinations thereof.
In yet another embodiment, the present invention provides methods of inducing phase II enzymes by providing wasabi root fiber powder, artichoke leaf extract, asparagus extract, kudzu root extract, oregano extract, schisandra berry extract, notoginseng (ethanol extract of Panax notoginseng root), sanchi (water extracts from Panax notoginseng root), Gegen (Pueraria omeiensis), spinach dehydrate, or combinations thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a bar graph showing the effect of various formulations in protecting the liver from ethanol insults as measured by MTT.
Figure 2 is a bar graph showing the effect of various formulations in protecting the liver from ethanol insults as measured by LDH. Figure 3 is a bar graph showing the effect of various formulations in protecting the liver from CCl4 insults as measured by MTT.
Figure 4 is a bar graph showing the effect of various formulations in protecting the liver from CCl4 insults as measured by LDH.
DETAILED DESCRIPTION OF THE INVENTION
It is to be understood that this invention is not limited to the particular methodology or protocols described herein. Further, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which will be limited only by the claims.
Ingredients for Use in Formulations of the Present Invention
The present invention is based on the surprising discovery that unique combinations of the following ingredients, described more folly in Table 1, improve liver health: wasabi root fiber powder, artichoke leaf extract, asparagus extract, kudzu root extract, oregano extract, schisandra berry extract, notoginseng (ethanol extract of Panax notoginseng root), sanchi (water extracts from Panax notoginseng root), Gegen (Pueraria omeiensis), and spinach dehydrate.
More specifically, the formulations of the present invention improve liver health by protecting the liver against alcohol and carbon tetrachloride insults. Additionally, the formulations improve liver health by inducing phase II enzymes. Phase II enzymes are responsible for removing potential carcinogens by aiding in their removal from the body.
"Protecting the liver against alcohol insults" and "protecting the liver against carbon tetrachloride insults" refers to the ability of a formulation described herein to preserve or improve existing liver function.
Table 1
Figure imgf000004_0001
Figure imgf000006_0001
Formulations of the Present Invention
Table 2 illustrates representative daily amounts of the ingredients that can be included in the supplement.
Table 2
Figure imgf000006_0002
EXAMPLES
The following are illustrative examples of formulations made into tablets according to this invention and it should be understood that they do not limit the scope of the invention. Example 1
Figure imgf000007_0001
Figure imgf000008_0001
Figure imgf000009_0002
Figure imgf000009_0003
Figure imgf000010_0001
Figure imgf000010_0002
Figure imgf000010_0003
The above exemplary tabletted formulations can be manufactured according to typical methods known in the industry. For example, wasabi root fiber powder, artichoke leaf extract, schizandra berry extract, notoginseng and spinach dehydrate are passed through a SWECO separator equipped with a 20 mesh screen into a 100 cubic foot PK blender. Microcrystalline cellulose is added to the blend in the PK blender. The ingredients are blended for ten minutes. Cellulose gum and silicon dioxide are passed through a SWECO separator equipped with a 20 mesh screen directly into the 100 cubic foot PK blender. The ingredients are blended for ten minutes. Next, stearic acidis passed through a SWECO separator equipped with a 20 mesh screen directly into the 100 cubic foot PK blender. The mixture is blended for an additional five minutes. The resulting mixture is discharged into totes or supersacks, and compressed into tablets.
Methods of Administration Formulations of the present invention may be formulated in an acceptable carrier and may be prepared, packaged, and labeled for promoting health, liver function, protecting against alcohol and/or chemical insults to the liver, and/or inducing phase II enzymes to promote healthy liver function. The formulations of the present invention and their acceptable carriers may be formulated for oral administration in the form of a pill, tablet, dried or powdered product for reconstitution with water or other suitable vehicle before use, bar, food, solution, syrup, suspension, beverage, lozenge, etc. The formulations of the present invention may also be parenterally administered or administered by inhalation or insufflation (either through the mouth or nose).
Liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). When administered in the form of a beverage, formulations of the present invention may be water-based, milk-based, tea-based, fruit juice-based, or some combination thereof.
Formulations of the present invention may also be orally administered in the form of a solid prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose; fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., cellulose gum, potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Formulations of the present invention that are orally administered can further comprise thickeners, including xanthum gum, carboymethyl-cellulose, carboxyethylcellulose, hydroxypropylcellulose, methylcellulose, microcrystalline cellulose, starches, dextrins, fermented whey, tofu, maltodextrins, polyols, including sugar alcohols (e.g., sorbitol and mannitol), carbohydrates (e.g. lactose), propylene glycol alginate, gellan gum, guar, pectin, tragacanth gum, gum acacia, locust bean gum, gum arabic, gelatin, as well as mixtures of these thickeners.
Orally administered formulations of the present invention can contain an effective amount of one or more sweeteners, including carbohydrate sweeteners and natural and/or artificial no/low calorie sweeteners. The amount of the sweetener used in the formulations of the present invention will vary, but typically depends on the type of sweetener used and the sweetness intensity desired.
In addition to the formulations described previously, the compounds may also be a formulated as a sustained and/or timed release formulation. The formulations must be maintained above some minimum therapeutic dose to be effective. Common timed and/or controlled release delivery systems include, but are not be restricted to, starches, osmotic pumps, or gelatin micro capsules.
The formulations may, if desired, be presented in a pack or dispenser device which may comprise one or more unit dosage forms comprising a formulation of the present invention. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration.
Other useful dosage forms can be prepared by methods and techniques that will be well understood by those of skill in the art and may include the use of additional ingredients in producing tablets, capsules, or liquid dosage forms. The dose, and dose frequency, will vary according to the age, body weight, condition and response of the individual consumer or patient, and the particular formulation of the present invention that is used.
It is intended that the foregoing detailed description be regarded as illustrative rather than limiting. The present invention is further illustrated by the following experimental investigations and examples, which should not be construed as limiting.
Bioassay Study on Individual Ingredients
Several materials were submitted for bioassay testing to try to predict their protective ability against alcohol and chemically induced liver damage. To assess this, human liver cells were treated with the material and an insult, and cell viability was measured using two different assays. Alcohol damage was mimicked using 2.5% ethanol as the insult. CCl4 at 0.2% was used for the chemical insult. These concentrations were chosen because they were the concentrations that produced 20% cell death in preliminary experiments, and would therefore not cause the cells to undergo irreversible cell death/damage.
Materials were tested at three concentrations (1, 10 and 100 μg/mL) and an estimation of the EC-50 was determined (see Experimental section below for more details). In addition to the cell viability assays, phase II enzyme induction testing was also conducted to determine if a material had detoxification properties. The phase II enzyme induction assay measures a sample's ability to induce quinone reductase (a phase II enzyme) which is indicative of detoxification events. Phase II enzymes are responsible for removing potential carcinogens by aiding in their removal from the body. Broccoli is known as a good phase II enzyme inducer because of its sulforaphane content. Pure sulforaphane has activity at 106 U/g, while broccoli has reported activity from 4000 - 74;000 U/g (dependent on variety, form, and/or extraction conditions). Activity above 30,000 U/g is considered excellent. Activity less than 5000 U/g is considered minimal. Most materials that are considered good phase II enzyme inducers will have activity between 15,000-30,000 U/g.
Tables 3 and 4 summarize the EC-50 values of all materials tested. Table 3 is specifically for results of the ethanol protection, and Table 4 for carbon tetrachloride. Table 5 gives the results of the phase II enzyme induction assay.
Table 3:' EC-50 results of the MTT and LDH assays for Ethanol protection assessment.
Figure imgf000013_0001
Figure imgf000014_0001
Figure imgf000015_0001
Table 4: EC-50 results of the MTT and LDH assays for CC14 protection assessment.
Figure imgf000015_0002
Figure imgf000016_0001
Figure imgf000017_0001
* Inducer units per gram of fresh weight of material. - = Negligible activity, + = Little activity,
++ = Good activity, +++ = Very good activity, ++++ = Excellent activity. **Not reportable due to toxicity to cells. Experimental
For the ethanol protection and CCl4 assessments, stock sample solutions are made in DMSO, then diluted in cell culture media for testing. Treatment of HepG2 cells (human liver cell line) is done by adding 100 μL of sample to each of three wells of a 96-well microtiter plate. After a 4 hour incubation, the insult is added (2.5% ethanol or 0.2% carbon tetrachloride) and an additional overnight incubation period is conducted. The next day, cell viability is measured using two different assays. First, using the CytoTox-ONE Homogenous Membrane Integrity Assay by Promega, the number of non-viable cells is estimated by measuring the release of LDH into the media. LDH leaks out of the cell when the cell membrane is compromised. The second assay is the MTT assay, which measures the reduction of a yellow tetrazolim salt into an insoluble purple formazen product by the mitochondria of viable cells. Following an incubation with the MTT solution, isopropanol is added to solubilize the colored crystals. The amount of color produced is directly proportional to the number of viable cells. For the phase II enzyme induction assay, stock sample solutions are made in acetonitrile, then diluted in cell culture media for testing. Treatment of Hepalclc7 cells (murine hepatoma cell line) is done by adding 150μL of sample to each of three wells, in a 96-well microtiter plate. After 48 hours incubation, induction activity of quinone reductase is established by measuring the NADPH-dependent, menadiol-mediated reduction of MTT. Activity is reported as inducer units per gram of fresh weight of material, where one unit of inducer activity is defined as the amount of inducer required to double the quinone reductase specific activity of Hepalclc7 cells. Bioassay Study on Ingredient Combinations
Several blends of liver detoxification ingredients were submitted for bioassay testing to try to predict their protective ability against alcohol and chemically induced liver damage. These blends were tested alongside two products already on the market for liver health - NUTRILITE® Milk Thistle and Dandelion and China's King Drink. To assess this, human liver cells were treated with the sample and an insult, and cell viability was measured using two different assays. Alcohol damage was mimicked using 2.5% ethanol as the insult. CCl4 at 0.2% was used for the chemical insult. These concentrations were chosen because they were the concentrations that produced 20% cell death in preliminary experiments, and would therefore not cause the cells to undergo irreversible cell death/damage. Samples were tested at three concentrations (1, 10 and 100 μg/mL) and an estimation of the EC-50 was determined (see Experimental section below for more details). Materials with EC-50 values at or below lOμg/mL are indicative of being the most efficacious, assuming 10% absorption of the material in 5L of blood (average human volume).
The blends that showed the greatest efficacy against CCl4 liver cell damage were 8523- 25-CI (Example 1), 8523-27-CI (Example 8), 8523-28-CI (Example 2), and 8523-30-CI (Example 3). Other blends that performed well (exhibited 40% protection at <10 μg/mL) were 8523-20-CI, 8523-22-CI, 8523-24-CI, 8523-26-CI and 8523-31 -CI. None of the blends showed efficacy at < 10 μg/mL against ethanol liver cell damage. The control products (NUTRILITE milk thistle and King Drink) also did not achieve EC-50 values of <10 μg/mL. The blends that had EC-50 values >100 μg/mL were 8523-28-CI (Example 2), 8523-30-CI (Example 3), 8523-31 -CI (Example 4) and 8523-32-CI (Example 5). Tables 6-9 summarize the results of all the samples tested. These results are also seen in Figures 1-4.
Table 6: Results of the MTT assays for ethanol protection assessment (* = achieved EC-40 at one or more concentration)
Figure imgf000019_0001
Table 7: EC-50 results of the LDH assays for ethanol protection assessment. (* = achieved EC-40 at one or more concentration)
Figure imgf000020_0001
Table 8: EC-50 results of the MTT assays for CC14 protection assessment. (* = achieved EC-40 at one or more concentration)
Figure imgf000020_0002
Table 9: EC-50 results of the LDH assays for CC14 protection assessment.
Figure imgf000021_0001
In addition to the cell viability assays, phase II enzyme induction testing was also conducted to determine if a material had detoxification properties. The phase II enzyme induction assay measures a sample's ability to induce quinone reductase (a phase II enzyme) which is indicative of detoxification events. Broccoli is known as a good phase II enzyme inducer because of its sulforaphane content. Pure sulforaphane has activity at 106
U/g, while broccoli has reported activity from 4000 - 74,000 U/g (dependent on variety, form, and/or extraction conditions). Activity above 30,000 U/g is considered excellent.
Activity less than 5,000 U/g is considered minimal. Most materials that are considered good phase II enzyme inducers will have activity between 15,000-30,000 U/g.
All the blends tested had good to excellent phase II enzyme induction activity. The highest activity came from 8523-27 (Example 8) and the lowest from 8523-31 (Example 4). Excellent activity also came from 8523-22, 8523-23, 8523-30 (Example 3), 8523-32
(Example 5) and 8523-33. Table 10 gives the results of the phase II enzyme induction assay for all samples tested. Table 10: Phase II Enzyme (Quinone Reductase) Induction Assay Results
Figure imgf000022_0001
* Inducer units per gram of fresh weight of material. - = Negligible activity, + = Little activity,
++ = Good activity, +++ = Very good activity, ++++ = Excellent activity. **Not reportable due to toxicity to cells.
Experimental Stock sample solutions are made in DMSO, then diluted in cell culture media for testing. Treatment of HepG2 cells (human liver cell line) is done by adding 100 μL of sample to each of three wells of a 96-well microtiter plate. After a 4 hour incubation, the insult is added (2.5% ethanol or 0.2% carbon tetrachloride) and an additional overnight incubation period is conducted. The next day, cell viability is measured using two different assays. First, using the CYTOTOX-ONE™ Homogenous Membrane Integrity Assay by Promega, the number of non- viable cells is estimated by measuring the release of LDH into the media. LDH leaks out of the cell when the cell membrane is compromised. The second assay is the MTT assay, which measures the reduction of a yellow tetrazolim salt into an insoluble purple formazen product by the mitochondria of viable cells. Following an incubation with the MTT solution, isopropanol is added to solubilize the colored crystals. The amount of color produced is directly proportional to the number of viable cells. Protection is determined by first calculating a percent toxicity of each well (1- experimental/negative control), after averaging the three replicates. Percent protection is then calculated by the following: (%Toxicity Positive Control - % Toxicity Sample)/% Toxicity Positive Control, the positive control being either 2.5% ethanol or 0.2% carbon tetrachloride. The concentration that exhibits 50% protection (EC-50) can then be assessed. For the purpose of this experiment, this was categorized as either <1, 1, 1-10, 10, 10-100, 100 or >100 μg/mL.
For the phase II enzyme induction assay, stock sample solutions are made in acetonitrile, then diluted in cell culture media for testing. Treatment of Hepalclc7 cells (murine hepatoma cell line) is done by adding 150μL of sample to each of three wells, in a 96-well microtiter plate. After 48 hours incubation, induction activity of quinone reductase is established by measuring the NADPH-dependent, menadiol-mediated reduction of MTT. Activity is reported as inducer units per gram of fresh weight of material, where one unit of inducer activity is defined as the amount of inducer required to double the quinone reductase specific activity of Hepal cl c7 cells.
Mammalian Studies
Clinical testing can be conducted to confirm the efficacy of the formulations on liver health. It is expected that the formulations will improve liver health by protecting the liver from chemical and alcohol insults. A protocol for such testing follows. PROTOCOL 1 : CCl4 LIVER INJURY MODEL 1.1 Principles.
When CCl4 is activated by microsomal enzyme of liver, trichloromethane free radicals (CCl3-) are formed. Covalent combination of this free radical with protein results in impairment of protein synthesis and disorder of lipid catabolism, causing accumulation of triglyceride (TG) in liver cells. CCl3- also can combine with O2 rapidly to form trichloromethane peroxide free radicals (CCl3O2-), leading to lipid peroxidation which causes degenerative injury of cell membrane, leakage of enzymes and various types of pathological changes of cells and even necrosis. 1.2 Experimental animals.
Adult rats or mice of single sex. Each group consists of 8-12 rats (180-22Og) or 10- 15 mice (18-22g). 1.3 Experimental methods and procedures. 1.3.1 Dosage groups and duration of administration of the test sample.
Three dosage groups, one blank control group and one model control group are set. The dosage of one of the dosage groups is 10 times (mice) or 5 times (rats) the recommended human dosage. CCl4 (analytically pure) is used to form liver injury model. The method of forming model can use intragastric administration or intraperitoneal injection. The concentration of CCl4 for intragastric administration in mice is 1%. CCl4 is diluted with edible vegetable oil and the dosage for intragastric administration is 5mL/kg BW (the dosage in terms of CCl4 is 80mg/kg BW). The concentration of CCl4 for intragastric administration in rats is 2%-3% and the dosage is 5mL/kg BW (the dosage in terms of CCl4 is 160-240mg/kg BW). Positive control group and solvent control group may be set if necessary. The duration of administration of the test sample is 30 days and can be prolonged to 45 days if necessary.
1.3.2 Route of administration of the test sample.
The test sample is given intragastrically. If this is impossible, the test sample can be mixed into the feed or drinking water and the feed intake or water drunk is recorded.
1.3.3 Experimental procedures.
The animals of the experimental group are given intragastrically the test sample daily, while those of the blank control group and the model control group are given distilled water. The animals are weighed twice a week for adjusting the dosage of the test sample. On the eve of day 30 of the experiment, the animals of various groups fast for 16h. The animals of the model group and various test sample groups are given intragastrically single dose of CCl4, while those of the blank control group are given vegetable oil. The animals of the experimental group continue to receive the test sample until the end of the experiment (the interval between administration of the test sample and CCl4 is over 4h). After giving CCl4, the animals are sacrificed 24h or 48h later according to the actual conditions. Blood is taken and serum is separated for measuring ALT and AST. Liver is taken for histopathological examination.
1.3.4 Indices for measurement.
Serum glutamate-pyruvate transaminase (ALT), glutamic-oxaloacetic transaminase (AST), histopathological examination of liver. 1.4 Measurement of ALT and AST. 1.4.1 Measuring method.
Full-automatic biochemical analyzer or Reitman-Frankel method (reagent kit) can be selected. 1.4.1 Data treatment and result assessment.
Variance analysis is used, but variance homogeneity test should be performed first according to the procedures of variance analysis. If the variance is homogeneous, F value is calculated. If F value is < Fo.o5, the conclusion is that the difference between means of different groups is not significant. If F value is > Fo.O5 and P is < 0.05, the method of paired comparison of means between several experimental groups and one control group is used for statistical analysis. For data with abnormal distribution or variance inhomogeneity, appropriate conversion of variables is performed and the converted data are used for statistical analysis after the requirement of normal or variance homogeneity is fulfilled. If the purpose of normal or variance homogeneity is still not achieved after conversion of variables, rank test is used for statistical analysis.
If ALT and AST of the test sample group are different significantly from those of the model control group, the results of ALT and AST can be assessed as positive respectively. 1.5 Histopatholofiical changes of liver, diagnostic criteria and result assessment. 1.5.1 Experimental materials.
The left lobe of rat liver is fixed with 10% formalin. The liver tissue is taken from the cross section of middle part of the left lobe of liver for routine preparation of pathological section (paraffin embedding, H.E. strain). 1.5.2 Microscopic examination.
Using the 40-fold objective to observe continuously the whole tissue section, the pathological changes of cells are recorded beginning from the visual field of one end of the liver. The degenerative changes of central liver cells of the lobe and necrosis of a few cells can be seen. The main types of pathological changes are ballooning degeneration, fatty degeneration, condensation of cytoplasm, hydropic degeneration and necrosis of liver cells, etc.
1.5.3 Criteria for rating.
Each pathological change accounting for the portion of area of visual field in each visual field is recorded respectively and the total score of pathological changes in the visual fields observed is added up.
Ballooning degeneration of liver cells: (swelling of cells, a little cytoplasm remains)
Roughly normal 0 points
Liver cells with ballooning degeneration account for 1/4 of whole visual field 1 point Liver cells with ballooning degeneration account for 1/2 of whole visual field 2 points
Liver cells with ballooning degeneration account for 3/4 of whole visual field 3 points
Liver cells with ballooning degeneration account for whole visual field 4 points
Fatty degeneration of liver cells: (distinctly demarcated fat drop vacuoles appear in cytoplasm of liver cells)
Roughly normal , 0 points
Liver cells with fatty degeneration account for 1/4 of whole visual field 1 point Liver cells with fatty degeneration account for 1/2 of whole visual field 2 points
Liver cells with fatty degeneration account for 3/4 of whole visual field 3 points
Liver cells with fatty degeneration account for whole visual field 4 points
Condensation of cytoplasm: (eosinophilic stain is enhanced)
Roughly normal 0 points
Liver cells with condensation of cytoplasm account for 1/4 of whole visual field 1 point
Liver cells with condensation of cytoplasm account for 2/4 of whole visual field 2 points
Liver cells with condensation of cytoplasm account for 3/4 of whole visual field 3 points Liver cells with condensation of cytoplasm account for whole visual field 4 points
Hydropic degeneration:
No liver cell with hydropic change is seen 0 points Liver cells with hydropic degeneration account for 1/4 of whole visual field 1 point
Liver cells with hydropic degeneration account for 2/4 of whole visual field 2 points
* A
Liver cells with hydropic degeneration account for 3/4 of whole visual field 3 points
Diffuse liver cells with hydropic degeneration account for whole visual field 4 points
Necrosis of liver cells: (eosinophilic change of cytoplasm, coagulation necrosis)
No necrotic cell is seen 0 points
Sporadic necrotic cells account for 1/4 of whole visual field 1 point
Necrotic cells account for 2/4 of whole visual field 2 points Necrotic cells account for 3/4 of whole visual field 3 points
Diffuse necrotic cells account for whole visual field 4 points
1.5.4 Data treatment and result assessment. Variance analysis is used, but variance homogeneity test should be performed first according to the procedures of variance analysis. If the variance is homogeneous, F value is calculated. If F value is < Fo.os, the conclusion is that the difference between means of different groups is not significant. IfF value is > F0.05 and P is < 0.05, the method of paired comparison of means between several experimental groups and one control group is used for statistical analysis. For data with abnormal distribution or variance inhomogeneity, appropriate conversion of variables is performed and the converted data are used for statistical analysis after the requirement of normal or variance homogeneity is fulfilled. If the purpose of normal or variance homogeneity is still not achieved after conversion of variables, rank test is used for statistical analysis. Among the pathological changes of liver cells including ballooning degeneration, fatty degeneration, cytoplasm condensation , hydropic degeneration and necrosis of liver cells, if the necrosis of liver cells in any dosage group of test sample is alleviated as compared with that in the model control group with significant difference and other types of pathological changes are alleviated significantly or have no significant difference as compared with those in the model control group, the results of animal pathological experiment can be assessed as positive.
If aggravation and alleviation of the 4 types of pathological changes of liver cells, namely, ballooning degeneration, fatty degeneration, cytoplasm condensation and hydropic degeneration, are present simultaneously With significant difference and necrosis of liver cells is alleviated in any one dosage group of the test sample with significant difference as compared With the model control group, the scores of various pathological changes and double of necrosis score are added together. The total score is used for statistical analysis. If the total score has significant difference, the results of animal pathological experiment can be assessed as positive. 1.6 Assessment of results.
It is expected that any one of the two blood biochemical indices, ALT and AST, and the result of pathological examination will be positive and the test sample will be assessed as assisting in the protection against chemical injury to the liver. PROTOCOL 2: MODEL OF ALCOHOLIC INJURY OF LIVER 2.1 Principles.
After taking large amounts of ethyl alcohol, massive dehydroxylation catalyzed by ethanol dehydrogenase causes disorder of tricarboxylic acid cycle and weakening of oxidation of fatty acid, thereby influencing fat metabolism and precipitation of fat in liver cells. At the same time, ethyl alcohol can activate oxygen molecules and cause production of oxygen free radicals, leading to lipid peroxidation of liver cell membrane and depletion of reduced glutathione in body.
2.2 Experimental animals.
Adult mice or rats of single sex. Each group consists of 8-12 rats (180-22Og) or 10- 15 mice (18-22g).
2.3 Experimental methods and procedures.
2.3.1 Dosage groups and duration of administration of the test sample.
Three dosage groups, one blank control group and one model control group are set. The dosage of one of the dosage groups is 10 times (mice) or 5 times (rats) the recommended human dosage. A positive control group may be set if necessary. Absolute ethyl alcohol (analytically pure) is used to form model of liver injury. The concentration of absolute ethyl alcohol is 50% (diluted with distilled water) and the dosage for intragastric administration to mice is 12-14mL/kg BW (equivalent to ethyl alcohol 6000-7000mg/kg BW). The duration of administration of the test sample is 30 days and can be prolonged to 45days if necessary.
2.3.2 Route of administration of the test sample.
The test sample is given intragastrically. If intragastric administration is impossible, the test sample can be mixed in feed or drinking water, and the feed intake and drinking water drunk of each animal is recorded. 2.3.3 Experimental procedures.
The animals of the test sample groups are given intragastrically the test sample every day and those of the blank control group and model control group are given distilled water.
The animals are weighed twice weekly and the dose of the test sample is adjusted according to body weight. At the time of completion of administration of the test sample, a single dose of 50% ethyl alcohol 12mL/kg BW is given to the animals of the model control group and three dosage groups, while the animals of the blank control group are given distilled water.
After fasting for 16h, the animals are sacrificed for examination of various indices and histopathological examination. 2.3.4 Indices for examination.
Malondialdehyde (MDA), reduced glutathione (GSH), triglyceride (TG) content of liver.
2.4 Method for measuring the degradation product of lipid peroxide malondialdehyde (MP A) in liver homogenate.
2.4.1 Principle.
MDA is one of the final products of peroxidation of lipids of cell membrane. Measuring MDA content can estimate indirectly the degree of lipid peroxidation. When MDA and thiobarbital are heated together in acidic condition, pink-colored complex is formed and its absorption peak is at 535nm, from which the MDA content can be measured.
2.4.2 Instruments and reagents.
Instruments: 721 spectrophotometer, sample micro-applicator, thermostat water bath, ordinary centrifuge, mixing rotator, centrifuge tube with stopper, tissue homogenizer.
Reagents: 0.2M acetate buffer solution, pH 3.5: 0.2M acetic acid solution 185mL
0.2 M sodium acetate solution 15mL lmmol/L tetraethoxyl propane (stock solution, kept at 4°C for 3 months), diluted with water to 40nmol/mL just before use:
8.1% sodium dodecyl sulfate SDS 0.8% thiobarbital TBA
0.2 M phosphate buffer solution, pH 7.4
0.2 M disodium hydrogen phosphate 192OmL 0.2 M potassium dihydrogen phosphate 48OmL
2.4.3 Experimental procedures. 2.4.3.1 Preparation of sample.
Tissue homogenate sample: certain quantity of the organ needed is rinsed with normal saline, wiped to dry, weighed, minced and put into homogenizer. 0.2 M phosphate buffer solution is added and the mixture is homogenized at 2000r/min for 10s. The centrifugation is repeated 3 times with 30s intervals to form 5% tissue homogenate (WfV). The homogenate is centrifugalized at 3000r/min for 5-10min and the supernatant is taken for measurement.
2.4.3.2 Measurement of the sample.
Reagent Blank tube Sample tube Standard tube 5% tissue homogenate O.lmL
40nmol/mL tetraethoxyl propane O. ImL
8.1% SDS 0.2mL 0.2mL 0.2mL
0.2 M acetate buffer solution 1.5mL 1.5mL 1.5mL
0.8% TBA 1.5mL 1.5mL 1.5mL
H2O 0.8mL 0.7mL 0.7mL
Mix to homogenize, boiling water bath for 60min protect from light, cooled with flowing water, colorimetry at 532nm : =
2.4.3.3 Calculation.
B - A B - A 1 Lipid peroxide content (nmol/mg tissue)= xCxK= x40x F - A F - A 0.05x1000
Lipid peroxide content (nmol/lOOmg protein) = B - A B - A 1 1
Figure imgf000030_0001
F - A F - A 0.05 Protein (mg)/g tissue A: absorbance of blank tube
B: absorbance of sample tube F: absorbance of tetraethoxyl propane C: concentration of tetraethoxyl propane (40nmol/rnL) K: multiple of dilution 2.4.3.4 Data treatment and result assessment.
The data are analyzed with variance analysis, but variance homogeneity test should be performed first according to the procedures of variance analysis. If the variance is homogeneous, F value is calculated. If F value is < F0.05, the conclusion is that the difference between means of different groups is not significant. IfF value is > Fo.os and P is < 0.05, the method of paired comparison of means between several experimental groups and one control group is used for statistical analysis. For data with abnormal distribution or variance inhomogeneity, appropriate conversion of variables is performed and the converted data are used for statistical analysis after the requirement of normal or variance homogeneity is fulfilled. If the purpose of normal or variance homogeneity is still not achieved after conversion of variables, rank test is used for statistical analysis.
Assessment of results
It is expected that the MDA content of test sample groups will be significantly different from that of the model control group and, as such, the result of this index will be assessed as positive. 2.5 Method for measuring reduced glutathione (GSH) in liver homogenate.
2.5.1 Principle.
Reaction between GSH and 5,5'-dithionitroformic acid (DTNB) catalyzed by GSH- Px produces yellow-colored 5-thio-2-nitro-formic acid anion which has maximum absorption peak at wavelength of 423nm. Measuring the concentration of this ion can calculate GSH content.
2.5.2 Reagents. 0.9% normal saline
4% sulfosalicylic acid solution 0.1mol/L PBS solution (pH = 8.0) Na2HPO4 13.452g
KH2PO4 0.722g Distilled water ad 100OmL.
0.004% DTNB solution: DTNB 40mg is dissolved in 100OmL of 0.1mol/L PBS solution (pH = 8.0). Sodium azide buffer solution.
NaN3 16.25mg
EDTA-Na2 7.44mg
Na2HPO4 1.732g
NaH2PO4 1.076g Distilled water ad 100OmL. Small amount of HCl and NaOH are used to regulate pH 7.0.
The solution is kept at 4°C. Standard solution: Reduced GSH 15.4mg is weighed and sodium azide buffer solution is added to 5OmL to make the final concentration lmmol/L. The solution is prepared just before use.
2.5.3 Methods.
5 2.5.3.1 Measurement of sample.
Normal saline 5mL is added to liver 0.5g. The mixture is well ground to form fine thick liquid (10% liver homogenate). After homogenizing, 4% sulfosalicylic acid 0.5mL is added to the homogenate 0.5mL. After mixing, the mixture is centrifugalized at 3000rpm for lOmin at room temperature and the supernatant is the sample.
-10
Reagent Tube for measurement Blank tube
Sample 0.5mL —
4% sulfosalicylic acid — 0.5mL
15 DINB 4.5mL 4.5mL
The mixture is mixed, laid aside for lOmin at room temperature and its absorbance is measured at 412nm.
2.5.3.2 Standard curve. 0
Reagent 1 2 3 4 5 6
lmmol/L GSH (mL) 0 0.05 0.10 0.15 0.20 0.25
Normal saline (mL) 0.50 0.45 0.40 0.35 0.20 0.25
25 DTNB (mL) 4.50 4.50 4.50 4.50 4.50 4.50
GSH content (μmol/L) 0 100 200 300 400 500
2.5.3.3 Calculation.
Sample GSH content (μmol/L liver tissue) = corresponding curve concentration 0 value (μmol/L) ÷50g/L
2.5.4 Data treatment and result assessment.
The data are analyzed with variance analysis, but variance homogeneity test is performed first according to the procedures of variance analysis. If the variance is homogeneous, F value is calculated. If F value is < Fo.o5, the conclusion is that the difference between means of different groups is not significant. If F value is > Fo.o5 and P is < 0.05, the method of paired comparison of means between several experimental groups and one control group is used for statistical analysis. For data with abnormal distribution or variance inhomogeneity, appropriate conversion of variables is performed and the converted data are used for statistical analysis after the requirement of normal or variance homogeneity is fulfilled. If the purpose of normal or variance homogeneity is still not achieved after conversion of variables, rank test is used.
Assessment of results
It is expected that the reduced GSH content of the test sample group will be significantly different from that of the model control group and, as such, the result of this index is assessed as positive.
2.6 Method for measuring triglyceride (TG) in liver homogenate. 2.6.1 Measuring method.
Triglyceride measurement reagent kit (glycerophosphoric acid oxidase peroxidase method) is used to measure the triglyceride content in 10% liver homogenate. Same as the method of measuring serum triglyceride, equal amount of 10% liver homogenate is used instead of serum and the measurement is performed according to the description of operation. The result of measurement is expressed as mmol/g liver weight. 2.6.2 Data treatment and result assessment.
The data are treated with variance analysis, but variance homogeneity test is performed first according to the procedures of variance analysis. If the variance is homogeneous, F value is calculated. If F value is < F0.05, the conclusion is that the difference between means of different groups is not significant. IfF value is > F0-05 and P is
≤ 0.05, the method of paired comparison of means between several experimental groups and one control group is used for statistical analysis. For data with abnormal distribution or variance inhomogeneity, appropriate conversion of variables is performed and the converted data are used for statistical analysis after the requirement of normal or variance homogeneity is fulfilled. If the purpose of normal or variance homogeneity is still not achieved after conversion of variables, rank test is used for statistical analysis. Assessment of results
It is expected that the TG of the test sample group will be significantly different from that of the model control group and, as such, the result of this index will be assessed as positive. 2.7 Histopathological changes of liver, diagnostic criteria and result assessment.
2.7.1 Experimental materials.
Cross section at middle part of left lobe of liver is performed for taking examination material. Frozen section is made and stained with Sudan III staining.
2.7.2 Microscopic examination. The pathological changes of the cells are recorded beginning from the visual field at one end of the liver. 40-fold objective is used for continuous observation of whole tissue section. The main objects for observation are distribution, range and area of fat drops in liver.
2.7.3 Criteria for rating. :
The fat drops in liver cells are sporadic and scarce 0 points
The liver cells containing fat drops do not exceed 1/4 1 point
The liver cells containing fat drops do not exceed 1/2 2 points
The liver cells containing fat drops do not exceed 3/4 3 points The liver tissue is almost replaced by fat drops 4 points
2.7.4 Data treatment and result assessment.
Variance analysis is used, but variance homogeneity test should be performed first according to the procedures of variance analysis. If the variance is homogeneous, F value is calculated. If F value. is < Fo.os, the conclusion is that the difference between means of different groups is not significant. IfF value is > F0.05 and P is < 0.05, the method of paired comparison of means between several experimental groups and. one control group is used for statistical analysis. For data with abnormal distribution or variance in homogeneity, appropriate conversion of variables is performed and the converted data are used for statistical analysis after the requirement of normal or variance homogeneity is fulfilled. If the purpose of normal or variance homogeneity is still not achieved after conversion of variables, rank test is used for statistical analysis.
It is expected that the fatty degeneration in any dosage group of test sample will be alleviated as compared with the model control group with statistical difference and, as such, the result will be assessed as positive. 2.8 Assessment of the results.
It is expected that the following conditions will be fulfilled and, as such, the test sample would be assessed as assisting in protection against alcoholic liver injury:
(a) The results of examination of 3 indices, namely, liver MDA, reduced GSH and TG3 are positive.
(b) Any two of the 3 indices, namely, liver MDA, reduced GSH and TG, are positive and the results of histopathological examination are positive.

Claims

WHAT IS CLAIMED:
1. A formulation comprising at least two ingredients selected from the group consisting of schisandra berry extract, notoginseng, kudzu root extract, spinach dehydrate, oregano extract, artichoke leaf extract, wasabi root powder, gegen root extract, sanchi, and asparagus dehydrate, wherein the formulation is effective for improving liver health.
2. The formulation of claim 1 wherein the schisandra berry extract is present in an amount from 150 mg - 1000 mg, the notoginseng is present in an amount from lOOmg - 500mg, the kudzu root extract is present in an amount from 150 mg - 1000 mg, the spinach dehydrate is present in an amount from 20 mg -500 mg, the oregano extract is present in an amount from 20 mg - 500 mg, the artichoke leaf extract is present in an amount from 200mg - lOOOmg, the wasabi root extract is present in an amount from 200 mg -1000 mg, the gegen root extract is present in an amount from 150 mg - 1000 mg, the sanchi is present in an amount from 100 mg 500 mg, and the asparagus dehydrate is present in an amount from 100mg- 500mg.
3. The formulation of claim 1 wherein the formulation comprises 150 mg-500 mg wasabi root powder, 200 mg - 1000 mg artichoke leaf extract, 150 mg - 1000 mg schisandra berry extract, 100 mg - 500 mg notoginseng, and 20 mg - 500 mg spinach dehydrate.
4. The formulation of claim 1 wherein the formulation comprises 150mg - 500mg wasabi, 200mg - lOOOmg artichoke leaf extract, 150 mg - lOOOmg kudzu root extract, lOOmg — 500mg notoginseng, and 20mg - 500mg oregano.
5. The formulation of claim 1 wherein the formulation comprises 200mg - lOOOmg artichoke leaf extract, 150 mg -1000 mg kudzu root extract, and 20 mg - 500 mg oregano extract.
6. A method of protecting the liver from carbon tetrachloride insults comprising providing the formulation of claim 1.
7. The method of claim 6 wherein the formulation comprises 150 mg — 500 mg wasabi root powder, 200 mg - lOOOmg artichoke leaf extract, 150 mg - 1000 mg schisandra berry extract, 100 mg - 500 mg notoginseng, and 20 mg - 500 mg spinach dehydrate.
8. The method of claim 6 wherein the at least two ingredients are selected from the group consisting of notoginseng, kudzu root extract, spinach dehydrate, oregano extract, wasabi root powder, gegen root extract, sanchi, and asparagus dehydrate.
9. The method of claim 6 wherein the formulation comprises wasabi and one of the ingredients selected from the group consisting of notoginseng, sanchi, kudzu root extract, gegen root extract, spinach, and oregano.
10. The method of claim 6 wherein the formulation is orally administered.
11. The method of claim 6 wherein the formulation is in the form of a tablet.
12. A method of protecting the liver from alcohol insults by providing the formulation of claim 1.
13. The method of claim 12 wherein the formulation comprises 150 mg - 500 mg wasabi root powder, 200 mg - lOOOmg artichoke leaf extract, 150 mg - 1000 mg schisandra berry extract, 100 mg - 500 mg notoginseng, and 20 mg - 500 mg spinach dehydrate.
14. The method of claim 12 wherein the formulation comprises artichoke and oregano.
15. The methods of claim 12 wherein the formulation is orally administered in the form of a tablet.
16. A method of inducing phase II enzymes comprising providing the formulation of claim 1.
17. The method of claim 16 wherein the formulation comprises 150 mg - 500 mg wasabi root powder, 200 mg - 1000 mg artichoke leaf extract, 150 mg - 1000 mg schisandra berry extract, 100 mg - 500 mg notoginseng, and 20 mg - 500 mg spinach dehydrate.
18. The method of claim 15 wherein the formulation comprises gegen root extract and one of the ingredients selected from the group consisting of artichoke leaf extract, wasabi root powder, oregano extract, notoginseng, sanchi, and kudzu root extract.
19. The method of claim 15 wherein the formulation is orally administered in the form of a tablet.
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